The present invention relates to a method for preparing phosphatidylinositol in high purity and high yield starting from a mixed phospholipid. The highly pure phosphatidylinositol obtained by the present invention can widely be used as an emulsifying agent in various fields such as foods, cosmetics, agricultural chemicals and fishery and also as an emulsifying agent or a liposome-forming basic material in medicines.
Phospholipids are widely present in living things such as animals, plants, microorganisms, algae or the like principally as constituent components of cell membranes thereof and play various important roles in the living bodies. Phospholipids derived from natural sources usually comprise a mixture of phosphatidylcholine, phosphatidylethanolamine, phosphatidylnositol, phosphatidylserine, phosphatidic acid, sphingomyelin or the like. To fractionate a specific phospholipid from the mixture, there have been used various methods, for instance, solvent-fractionation techniques such as extraction fractionation with a single solvent such as methanol, ethanol, isopropyl alcohol, hexane or chloroform and recrystallization from a mixed solvent; column chromatographic fractionation techniques in which an adsorbent such as silica gel, alumina or an ion-exchange resin is employed; and fractionation techniques through formation of a composite with CdCl.sub.2 or an acetylated derivative.
However, these solvent-fractionation techniques and the fractionation techniques through formation of derivatives cannot concentrate the phospholipid in a desired high purity. The combination of these methods with the column chromatographic fractionation techniques makes it possible to increase the purity of the resulting phospholipid, but the yield thereof is very low. For this reason, the thus-obtained product becomes very expensive and therefore the use thereof in an industrial scale is greatly limited. In particular, it is very difficult to isolate phosphatidylinositol from other components such as phosphatidylethanolamine and phosphatidic acid by the usual solvent-extraction method and highly purified phosphatidylinositol can be obtained only through a solvent-extraction method in which a variety of solvents are used and/or a column fractionation techniques in which a variety of solvents are used. However, in this case, the yield thereof is still very low.
On the other hand, it is also possible to chemically synthesize highly pure phospholipids and presently a variety of products chemically synthesized are industrially employed. Although chemical synthetic methods make it possible to finally prepare a single phospholipid, they suffer from various drawbacks, for instance, the deterioration and/or coloration of constitutive fatty acids or phosphoric acid esters having unsaturated bonds due to heat history during the synthesis thereof. Thus, in these methods, it is inevitable to use economically unfavorable and complicated processes for purification of the reaction products.
In addition to the foregoing methods, various attempts have been made to enzymatically convert phospholipids. For instance, Japanese Unexamined Published Patent Application (hereinafter referred to as "J. P. KOKAI") No. Sho 62-48390 discloses a method for preparing highly pure phosphatidylinositol which comprises the steps of treating a mixed phospholipid with phospholipase D derived from cabbage or rice bran to hydrolyze the phospholipid components other than phosphatidylinositol present in the mixture, extracting the reaction product with hexane and washing the extract with 5% acetic acid-containing ethanol. This method makes the best use of the characteristics of the enzyme in order to prepare phosphatidylinositol. Nevertheless, there has been a demand for the development of methods which make it possible to prepare phosphatidylinositol having a higher purity.